Acidithiobacillusspp. are the most active bacteria in bioleaching and bioremediation, because of their remarkable extreme environmental adaptabilities and unique metabolic characteristics. The researches on regulatory mechanisms of energy metabolism and stress resistance are critical for the understanding and application ofAcidithiobacillusspp. However, the lack of an ideal reporter gene has become an obstacle for studying genes expression and regulatory mechanism in these chemoautotrophic bacteria. In this study, we reported the firefly luciferase as a reporter gene forAcidithiobacillus caldus(A. caldus) and created a firefly luciferase (Luc) reporter system. The Luc system was applied for the quantitative analysis of the transcription strength of the promoters oftetHgene and thefeoAgene inA. caldus. Moreover, the regulating effect of ferric uptake regulator (Fur) on thefeoPgene inA. calduswas determined using the Luc system. The Luc reporter system is not only used in the study of regulatory mechanism ofA. caldus, but also applied in the researches of otherAcidithiobacillusspecies. Therefore, this study provides a new useful tool for the studies on the molecular biological mechanism and synthetic biological modification of these chemoautotrophic bacteria, which would promote the industrial application ofAcidithiobacillusspp.